Practical guide to diagnostic parasitology / Lynne S. Garcia.

Lynne Shore Garcia is the director of LSG & Associates, a firm providing training, teaching, and consultation services for diagnostic medical parasitology and health care administration. A former manager of the UCLA Clinical Microbiology Laboratory, she is a sought-after speaker (nationally and internationally) and author of hundreds of articles, book chapters, and books including two ASM Press books, Clinical Laboratory Management, Second Edition and Diagnostic Medical Parasitology, Sixth Edition

Includes bibliographical references and index.

TABLE OF CONTENTS
Preface xix

About the Author xxiii

Section 1 Philosophy and Approach to Diagnostic Parasitology 1

Neglected Tropical Diseases 2

Why Perform Diagnostic Parasitology Testing? 3

Travel 3

Population Movements 3

Control Issues 4

Climate Change 4

Epidemiologic Considerations 4

Compromised Patients; Potential Sex Bias Regarding Infection Susceptibility; Aging 4

Approach to Therapy 5

Who Should Perform Diagnostic Parasitology Testing? 5

Laboratory Personnel 5

Nonlaboratory Personnel 5

Where Should Diagnostic Parasitology Testing Be Performed? 6

Inpatient Setting 6

Outpatient or Referral Setting 6

Decentralized Testing 6

Physician Office Laboratories 6

Over-the-Counter (Home Care) Testing 7

Field Sites 7

What Factors Should Precipitate Testing? 7

Travel and Residence History 7

Immune Status of the Patient 7

Clinical Symptoms 7

Documented Previous Infection 7

Contact with Infected Individuals 8

Potential Outbreak Testing 8

Occupational Testing 8

Therapeutic Failure 8

What Testing Should Be Performed? 8

Routine Tests 8

Special Testing and Reference Laboratories 9

Specialized Referral Test Options—DPDx and Other Sites 9

Other (Nonmicrobiological) Testing 10

What Factors Should Be Considered in Development of Test Menus? 10

Physical Plant 10

Client Base 10

Customer Requirements and Perceived Levels of Service 10

Personnel Availability and Level of Expertise 10

Equipment 10

Budget 11

Risk Management Issues Associated with Stat Testing 11

Primary Amebic Meningoencephalitis 11

Granulomatous Amebic Encephalitis and Amebic Keratitis 14

Request for Blood Films 15

Automated Instrumentation 15

Patient Information 16

Conventional Microscopy 16

Table 1.1 Common features of the neglected tropical diseases 18

Section 2 Parasite Classification and Relevant Body Sites 19

Protozoa (Intestinal) 20

Amebae, Stramenopiles 20

Flagellates 22

Ciliates 22

Apicomplexa (Including Coccidia) 23

Microsporidia (Now Classified with the Fungi) 23

Protozoa (Other Body Sites) 24

Amebae 24

Flagellates 24

Apicomplexa (Including Coccidia) 24

Microsporidia (Now Classified with the Fungi) 25

Protozoa (Blood and Tissue) 26

Apicomplexa (Including Sporozoa) 26

Flagellates 26

Nematodes (Intestinal) 29

Nematodes (Tissue) 29

Nematodes (Blood and Tissue) 30

Cestodes (Intestinal) 31

Cestodes (Tissue) 32

Trematodes (Intestinal) 33

Trematodes (Liver and Lungs) 33

Trematodes (Blood) 35

Pentastomids 35

Acanthocephala 36

Table 2.1 Classification of human parasites 37

Table 2.2 Cosmopolitan distribution of common parasitic infections 39

Table 2.3 Body sites and possible parasites recovered 40

Section 3 Collection Options 43

Safety 44

Collection of Fresh Stool Specimens 44

Collection Method 44

Number of Specimens To Be Collected 45

Collection Times 47

Posttherapy Collection 47

Specimen Type, Stability, and Need for Preservation 47

Preservation of Stool Specimens 48

Overview of Preservatives 48

Formalin 49

Sodium Acetate-Acetic Acid-Formalin (SAF) 50

Schaudinn’s Fluid 51

Schaudinn’s Fluid containing PVA (Mercury Base) 52

Schaudinn’s Fluid containing PVA (Copper Base, Zinc Base) 52

Single-Vial Collection Systems (Other than SAF) 53

Universal Fixative (Total-Fix) 54

Quality Control for Preservatives 54

Procedure Notes for Use of Preservatives (Stool Fixative Collection Vials) 55

Procedure Limitations for Use of Preservatives (Stool Fixative Collection Vials) 55

Collection of Blood 56

Collection and Processing 56

Stat Test Requests and Risk Management Issues 57

Collection of Specimens from Other Body Sites 57

Table 3.1 Fecal specimens for parasites: options for collection and processing 58

Table 3.2 Approaches to stool parasitology: test ordering 61

Table 3.3 Preservatives and procedures commonly used in diagnostic parasitology (stool specimens) 62

Table 3.4 Advantages of thin and thick blood films 64

Table 3.5 Advantages and disadvantages of buffy coat films 64

Table 3.6 Potential problems of using EDTA anticoagulant for the preparation of thin and thick blood films 65

Table 3.7 Body sites and possible parasites recovered (trophozoites, cysts, oocysts, spores, adults, larvae, eggs, amastigotes, and trypomastigotes) 66

Section 4 Specimen Test Options: Routine Diagnostic Methods and Body Sites 69

Ova and Parasite Examination of Stool Specimens 70

Other Diagnostic Methods for Stool Specimens 72

Culture of Larval-Stage Nematodes 72

Estimation of Worm Burdens through Egg Counts 73

Hatching Test for Schistosome Eggs 73

Screening Stool Samples for Recovery of a Tapeworm Scolex 74

Testing of Other Intestinal Tract Specimens 74

Examination for Pinworm 74

Sigmoidoscopy Material 75

Duodenal Drainage Material 76

Duodenal Capsule Technique (Entero-Test) 76

Urogenital Tract Specimens 77

Sputum 77

Aspirates 78

Biopsy Specimens 78

Blood 79

Thin Blood Films 79

Thick Blood Films 80

Blood Staining Methods 80

Buffy Coat Films 80

QBC Microhematocrit Centrifugation Method 80

Knott Concentration 81

Membrane Filtration Technique 81

Culture Methods 81

Animal Inoculation and Xenodiagnosis 82

Antibody and Antigen Detection 82

Antibody Detection 82

Antigen Detection, Nucleic Acid-Based Tests, and Molecular Panels 84

Intradermal Tests 84

UV Autofluorescence 84

Table 4.1 Body sites, procedures and specimens, recommended methods and relevant parasites, and comments 86

Table 4.2 Serologic, antigen, and probe tests used in the diagnosis of parasitic infections 94

Section 5 Specific Test Procedures and Algorithms 95

Microscopy 96

CALIBRATION OF THE MICROSCOPE 96

Ova and Parasite Examination 99

DIRECT WET FECAL SMEAR 99

CONCENTRATION (Sedimentation and Flotation) 104

SEDIMENTATION CONCENTRATION (Formalin-Ethyl Acetate) 105

SEDIMENTATION CONCENTRATION USING THE UNIVERSAL FIXATIVE (Total-Fix) 109

FLOTATION CONCENTRATION (Zinc Sulfate) 110

PERMANENT STAINED SMEAR 114

Stains Used in the Permanent Stained Smear 116

TRICHROME STAIN (Wheatley’s Method) 116

IRON HEMATOXYLIN STAIN (Spencer-Monroe Method) 122

IRON HEMATOXYLIN STAIN (Tompkins-Miller Method) 127

MODIFIED IRON HEMATOXYLIN STAIN (Incorporating the Carbol Fuchsin Step) 128

CHLORAZOL BLACK E STAIN 131

Specialized Stains for Coccidia and Microsporidia 132

KINYOUN’S ACID-FAST STAIN (Cold Method) 132

MODIFIED ZIEHL-NEELSEN ACID-FAST STAIN (Hot Method) 136

CARBOL FUCHSIN NEGATIVE STAIN FOR CRYPTOSPORIDIUM (W. L. Current) 139

RAPID SAFRANIN METHOD FOR CRYPTOSPORIDIUM (D. Baxby) 139

RAPID SAFRANIN METHOD FOR CYCLOSPORA, USING A MICROWAVE OVEN (Govinda Visvesvara) 139

AURAMINE O STAIN FOR APLICOMPLEXA (INCLUDING COCCIDIA) (Thomas Hänscheid) 140

MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Weber, Green Counterstain) 144

MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Ryan, Blue Counterstain) 146

MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Evelyn Kokoskin, Hot Method) 149

Fecal Immunoassays for Intestinal Protozoa 150

ENTAMOEBA HISTOLYTICA 150

CRYPTOSPORIDIUM SPP. 151

GIARDIA LAMBLIA 151

KITS UNDER DEVELOPMENT 151

COMMENTS ON THE PERFORMANCE OF FECAL IMMUNOASSAYS 151

ENZYME IMMUNOASSAYS (Antigen Detection, No Centrifugation Recommended) 152

FLUORESCENCE (Visual Identification of the Organisms, Centrifugation Recommended) 152

LATERAL-FLOW CARTRIDGES (Antigen Detection, No Centrifugation Recommended) 153

Larval Nematode Culture 153

HARADA-MORI FILTER PAPER STRIP CULTURE 153

BAERMANN CONCENTRATION 155

AGAR PLATE CULTURE FOR STRONGYLOIDES STERCORALIS 158

Other Methods for Gastrointestinal Tract Specimens 161

EXAMINATION FOR PINWORM (Cellulose Tape Preparations) 161

SIGMOIDOSCOPY SPECIMENS (Direct Wet Smear) 163

SIGMOIDOSCOPY SPECIMENS (Permanent Stained Smear) 166

DUODENAL ASPIRATES 168

Methods for Urogenital Tract Specimens 171

RECEIPT OF DRY SMEARS 171

DIRECT SALINE MOUNT 171

PERMANENT STAINED SMEAR 174

URINE CONCENTRATION (Centrifugation) 176

URINE CONCENTRATION (Nuclepore Membrane Filter) 179

Preparation of Blood Films 181

THIN BLOOD FILMS 182

THICK BLOOD FILMS 184

COMBINATION THICK-THIN BLOOD FILMS 186

RISK MANAGEMENT ISSUES ASSOCIATED WITH BLOOD FILMS 187

USE OF A REFERENCE LABORATORY FOR PARASITE BLOOD DIAGNOSTIC TESTING 188

BLOOD FILM REPORTING WITH ADDITIONAL REPORT COMMENTS 189

BUFFY COAT BLOOD FILMS 189

Blood Stains 190

STAIN OPTIONS 190

GIEMSA STAIN 190

Blood Concentration 194

BUFFY COAT CONCENTRATION 194

KNOTT CONCENTRATION 195

MEMBRANE FILTRATION CONCENTRATION 197

Algorithm 5.1 Procedure for processing fresh stool for the O&P examination 201

Algorithm 5.2 Procedure for processing liquid specimens for the O&P examination 202

Algorithm 5.3 Procedure for processing preserved stool for the O&P examination—two-vial collection kit 203

Algorithm 5.4 Procedure for processing SAF-preserved stool for the O&P examination 204

Algorithm 5.5 Procedure for the use of Total-Fix (universal fixative, single-vial system) (this fixative contains no mercury, no PVA, and no formalin) 205

ALTERNATE METHOD FOR SMEAR PREPARATION DIRECTLY FROM VIAL 206

Algorithm 5.6 Use of various fixatives and their recommended stains 207

Algorithm 5.7 Ordering algorithm for laboratory examination for intestinal parasites 208

Algorithm 5.8 Procedure for processing blood specimens for examination 209

Table 5.1 Body sites, specimens, and recommended stains 210

Table 5.2 Approaches to stool parasitology: test ordering 214

Table 5.3 Laboratory test reports: notes and optional comments 215

Table 5.4 Parasitemia determined from conventional light microscopy: clinical correlation 216

Section 6 Commonly Asked Questions about Diagnostic Parasitology 217

Stool Parasitology 218

Specimen Collection 218

Specimen Processing 221

Diagnostic Methods 222

Stool Immunoassay Options 227

MOLECULAR TEST PANELS (FDA CLEARED) 229

A. APTIMA Trichomonas vaginalis Assay 230

B. Affirm VPIII Microbial Identification Test 231

C. Cepheid Xpert TV Assay for Trichomonas vaginalis from Men and Women 232

D. BD MAX Enteric Parasite Panel 233

E. BioFire FilmArray Gastrointestinal Panel 234

F. Luminex (Verigene II GI Flex Assay; Includes Parasites) 236

G. Other Pending Molecular Tests 238

Organism Identification 238

Reporting 242

Proficiency Testing 245

Tissues or Fluids 245

Blood 246

Specimen Collection 246

Specimen Processing 248

Diagnostic Methods 249

Organism Identification 254

Reporting 256

Proficiency Testing 257

General Questions 258

Section 7 Parasite Identification 265

Protozoa

Amebae (Intestinal) 266

Entamoeba histolytica 266

Entamoeba histolytica/Entamoeba dispar 270

Comments on Entamoeba moshkovskii and Entamoeba bangladeshi 273

Entamoeba bangladeshi 274

Entamoeba hartmanni 275

Entamoeba coli 278

Entamoeba gingivalis 281

Entamoeba polecki 283

Endolimax nana 284

Iodamoeba bütschlii 287

Blastocystis spp. (formerly Blastocystis hominis) 290

Flagellates (Intestinal) Giardia lamblia (G. duodenalis, G. intestinalis) 295

Dientamoeba fragilis 298

Chilomastix mesnili 302

Pentatrichomonas hominis 304

Enteromonas hominis, Retortamonas intestinalis 307

Ciliates (Intestinal) 310

Balantidium coli 310

Apicomplexa (Intestinal) 315

Cryptosporidium spp. 315

Coccidia (Intestinal) 319

Cyclospora cayetanensis 319

Cystoisospora (formerly Isospora) belli 323

Microsporidia (Intestinal) 326

Enterocytozoon bieneusi 326

Encephalitozoon intestinalis, Encephalitozoon spp. 329

Sporozoa (Blood and Tissue) 334

Plasmodium vivax 334

Plasmodium falciparum 339

Plasmodium malariae 343

Plasmodium ovale wallickeri, Plasmodium ovale curtisi 346

Plasmodium knowlesi 350

Malaria 353

Babesia spp. (Babesia microti, B. duncani, B. divergens, B. venatorum) 355

Toxoplasma gondii 359

Flagellates (Blood and Tissue) 368

Leishmania spp. 368

Trypanosoma brucei gambiense (West), T. brucei rhodesiense (East) 372

Trypanosoma cruzi 376

Amebae (Other Body Sites) 381

Naegleria fowleri 381

Acanthamoeba spp., Balamuthia mandrillaris, Sappinia diploidea 385

Flagellates (Other Body Sites) 389

Trichomonas vaginalis 389

NEMATODES 392

Intestinal 392

Ascaris lumbricoides 392

Trichuris trichiura, Capillaria philippinensis 395

Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum (Hookworms); Trichostrongylus spp. 398

Strongyloides stercoralis 401

Enterobius vermicularis 404

Tissue 407

Ancylostoma braziliense, Ancylostoma caninum, Uncinaria stenocephala (Dog and Cat Hookworms) 407

Toxocara canis, Toxocara cati (Dog and Cat Ascarid Worms) 410

Dracunculus medinensis 413

Trichinella spiralis 416

Blood and Tissue 419

Filarial Worms 419

CESTODES 425

Intestinal 425

Taenia saginata 425

Taenia solium 428

Diphyllobothrium latum 432

Hymenolepis (Rodentolepis) nana 435

Hymenolepis diminuta 438

Dipylidium caninum 440

Tissue 443

Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli,

Echinococcus oligarthrus 443

TREMATODES 448

Intestinal 448

Fasciolopsis buski 448

Liver and Lungs 451

Paragonimus westermani, Paragonimus mexicanus, Paragonimus kellicotti 451

Fasciola hepatica 454

Clonorchis (Opisthorchis) sinensis (Chinese liver fluke) 457

Blood 460

Schistosoma spp. (Schistosoma mansoni, S. haematobium, S. japonicum,

S. mekongi, S. malayensis, S. intercalatum) 460

Section 8 Common Problems in Parasite Identification 465

Table 8.1 Entamoeba sp. trophozoites versus macrophages 486

Table 8.2 Entamoeba sp. cysts versus PMNs 486

Table 8.3 Entamoeba histolytica versus Entamoeba coli precysts and cysts 487

Table 8.4 Endolimax nana versus Dientamoeba fragilis 488

Section 9 Identification Aids 489

DIAGNOSTIC CONSIDERATIONS 490

Table 9.1 Rapid diagnostic procedures 490

Table 9.2 Diagnostic characteristics for organisms in wet mounts (direct or concentration sediment) 491

Table 9.3 Diagnostic characteristics for organisms in permanent stained smears (e.g., Wheatley’s trichrome, iron-hematoxylin) 491

Identification Key 9.1 Identification of intestinal amebae (permanent stained smear) 491

Identification Key 9.2 Identification of intestinal flagellates 492

Identification Key 9.3 Identification of helminth eggs 493

Identification Key 9.4 Identification of microfilariae 494

PROTOZOA 495

Table 9.4 Intestinal protozoa: trophozoites of common amebae 495

Table 9.5 Intestinal protozoa: cysts of common amebae 497

Table 9.6 Intestinal protozoa: trophozoites of less common amebae 499

Table 9.7 Intestinal protozoa: cysts of less common amebae 499

Table 9.8 Morphologic criteria used to identify Blastocystis spp. 500

Table 9.9 Intestinal protozoa: trophozoites of flagellates 501

Table 9.10 Intestinal protozoa: cysts of flagellates 502

Table 9.11 Intestinal protozoa: ciliate (Balantidium coli) 503

Table 9.12 Apicomplexa 503

Table 9.13 Microsporidia (related to the Fungi): general information 504

Table 9.14 Microsporidia: recommended diagnostic techniques 505

Table 9.15 Comparison of Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, and Sappinia diploidea 506

Table 9.16 Characteristics of Trichomonas vaginalis 507

Table 9.17 Key characteristics of intestinal tract and urogenital system protozoa 508

HELMINTHS 512

Table 9.18 Normal life spans of the most common intestinal nematodes 512

Table 9.19 Characteristics of the most common intestinal nematodes 513

Table 9.20 Tissue nematodes 515

Table 9.21 Trichinella spiralis: life cycle stages and clinical conditions 517

Table 9.22 Characteristics of human microfilariae 518

Table 9.23 Characteristics of cestode parasites (intestinal) 519

Table 9.24 Tissue cestodes 521

Table 9.25 Characteristics of intestinal trematodes 523

Table 9.26 Characteristics of liver and lung trematodes 524

Table 9.27 Human paragonimiasis 526

Table 9.28 Characteristics of blood trematodes 527

Table 9.29 Key characteristics of helminths 528

BLOOD PARASITES 532

Table 9.30 Malaria characteristics with fresh blood or EDTA-blood 532

Table 9.31 Potential problems with using EDTA anticoagulant for the preparation of thin and thick blood films 533

Table 9.32 Plasmodia in Giemsa-stained thin blood smears 534

Table 9.33 Relevant issues for handling requests for identification of infectious blood parasites 537

Table 9.34 Features of human leishmanial infections 540

Table 9.35 Characteristics of American trypanosomiasis 541

Table 9.36 Characteristics of East and West African trypanosomiasis 541

Table 9.37 Key characteristics of blood parasites 541

Index 547

"As we move forward into the 21st century, the field of diagnostic medical parasitology continues to see some dramatic changes, including newly recognized pathogens, changing endemicity of familiar pathogens, disease control challenges, geographic and climate changes that support the spread of parasitic disease, new methodologies, expansion of diagnostic testing, and an ongoing review of the approach to and clinical relevance of this type of diagnostic testing on patient care within the managed care environment, as well as the world as a whole. The third edition of the Practical Guide to Diagnostic Parasitology is organized to provide maximum help to the user, particularly from the bench use perspective. New aspects of the field have been addressed in these sections, and many new figures and plates have been added, including extensive color images. Section 1 on the philosophy and approach to diagnostic parasitology has been expanded to include discussions on neglected tropical diseases, the impact of global climate change, population movements, potential outbreak testing, the development of laboratory test menus, and the risk management issues related to "stat" testing. The Section 2 discussion of organism classification and relevant tables has been expanded and updated to provide the user with current information related to changes in nomenclature and overall importance of the various parasite categories to human infection"-- Provided by publisher.

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